skMLCK基因对成肌细胞C2C12肌纤维组成及信号通路的影响  

Effects of skMLCK gene on muscle fiber composition and signaling pathway in C2C12 cells

作　　者：李孝涵 朱弘焱 韩佳琪 张尚松 田玉民 苏玉虹 LI Xiaohan;ZHU Hongyan;HAN Jiaqi;ZHANG Shangsong;TIAN Yumin;SU Yuhong(College of Animal Husbandry and Veterinary Medicine,Jinzhou Medical University,Jinzhou 121000,China;College of Basic Medical Sciences,Jinzhou Medical University,Jinzhou 121000,China;College of Food and Health,Jinzhou Medical University,Jinzhou 121000,China)

机构地区：[1]锦州医科大学畜牧兽医学院,辽宁锦州121000 [2]锦州医科大学基础医学院,辽宁锦州121000 [3]锦州医科大学食品与健康学院,辽宁锦州121000

出　　处：《畜牧与兽医》2025年第3期47-57,共11页Animal Husbandry & Veterinary Medicine

基　　金：辽宁省种质创新藏粮于技专项(2023020531-JH1/102-04和JH1/102-05);辽宁省教育厅基础研究项目(LJKMZ20221240)。

摘　　要：旨在研究高、低表达骨骼肌肌球蛋白轻链激酶(skMLCK)基因对肌纤维组成成分和信号通路关键分子的影响。采用高表达和RNAi技术,获得skMLCK高、低表达稳转成肌细胞C2C12细胞株,提取细胞总RNA和总蛋白质,采用实时定量PCR和Western blot方法检测skMLCK高、低表达后C2C12中skMLCK基因,5种肌纤维组成成分肌球蛋白轻链(MLC)、肌球蛋白重链(MyHC)slow、MyHC 2a、MyHC 2x和MyHC 2b和6种信号通路关键因子肌球蛋白增强因子2C(MEF2C)、钙调蛋白(CaM)、促分裂原活化蛋白激酶p38(MAPKp38)、AMPK激活性蛋白激酶α2(AMPKα2)、RhoA和丝切蛋白2(CFL2)的表达情况。结果:在高、低表达稳转株中信号通路关键分子MAPKp38与skMLCK蛋白和mRNA表达量变化相同;2种肌纤维组成成分(MyHC 2x和MyHC 2b)和3种信号通路关键分子(CFL2、AMPKα2和RhoA)与skMLCK基因mRNA表达量变化相反;2种肌纤维组成成分(MyHC 2x和MyHC 2b)和4种信号通路关键分子(MEF2C、CaM、AMPKα2和CFL2)与skMLCK蛋白表达量变化相反,其余组分结果均一致;将所有蛋白质表达结果合并进行统计学分析发现,skMLCK基因与信号通路RhoA高度相关。综上,skMLCK基因可能通过改变C2C12细胞中RhoA信号通路来影响肌纤维的组成,skMLCK基因可以抑制CFL2的表达和肌纤维的生长。The aim of this study was to investigate the effects of high and low expressions of skMLCK on myofiber components and key molecules of the signaling pathway.The high and low expression and stable transformation of skMLCK into myoblast C2C12 cell line was obtained using the high expression and RNAi technology.Then,total RNA and total protein were extracted,and Real-time PCR and Western blot were used to detect skMLCK,five myofiber components(MLC,MyHC slow,MyHC 2a,MyHC 2x,and MyHC 2b),and six key molecules of the signaling pathways(MEF2C,CaM,MAPKp38,AMPKα2,RhoA and CFL2)in C2C12 cells after high and low skMLCK expressions.The results showed that key molecule MAPKp38 of the signaling pathway underwent the same changes with those og skMLCK protein and mRNA expression in high and low expression stable transplants.Two myofiber components(MyHC 2x and MyHC 2b)and the key molecules(CFL2,AMPKα2 and RhoA)of three signaling pathways showed opposite changes with mRNA expression of the skMLCK gene.Two myofiber components(MyHC 2x and MyHC 2b)and 4 signaling pathway key molecules(MEF2C,CaM,AMPKα2,and CFL2)were associated with the opposite changes in skMLCK protein expression;and the results of the remaining components were consistent.Statistical analysis of all protein expression results combined revealed that skMLCK was highly correlated with the signaling pathway RhoA.It suggested that skMLCK might affect myofiber composition by altering the RhoA signaling pathway in C2C12 cells.Meanwhile,skMLCK might inhibit CFL2 expression and myofiber growth.

关 键 词：骨骼肌肌球蛋白轻链激酶 C2C12细胞株 肌纤维 信号通路 

分 类 号：S852[农业科学—基础兽医学]